Nucleoside analogs are a class of compounds that have been shown to exert antiviral and antitumor activity both in vitro and in vivo, and thus, have been the subject of widespread research for the treatment of viral infections and cancer. Nucleoside analogs are molecules that antagonize or mimic the action of naturally occurring nucleosides. In general, nucleoside analogs consist of purine, pyrimidine or similar heterocyclic derivatives of adenine, cytosine, thymine, or guanine, and a glycoside or glycoside analog structure. Examples of nucleoside analogs include, but are not limited to, acyclovir, ribavirin, 3TC, AZT, araC, araA, DAPD ((-)-.beta.-D-2,6-diaminopurine dioxalane) and 5-FUDR. Several of these compounds are shown below. ##STR1##
Nucleoside analogs are therapeutically inactive compounds and are converted by host or viral enzymes to their respective active anti-metabolites, which, in turn, inhibit polymerases involved in viral or cell proliferation. The activation occurs by a variety of mechanisms, such as the addition of one or more phosphate groups and, or in combination with, other metabolic processes. For example, in the cell, the inosine of ddI is converted to adenine (ddA) and phosphate groups are then added to yield the active anti-metabolite ddATP.
Nucleoside analogs suffer from a variety of problems that limits their use in antiviral therapy.
1. Poor Solubility and Cell Penetration
Nucleoside analogs must be able to penetrate cell membranes and gain access to the intracellular space to be effective as therapeutics. Some nucleoside analogs traverse cell membranes by diffusional processes, which are governed by the charge and lipophilicity of the molecule. Others enter the cell by interaction with transporters for nucleosides present in the cell membrane. However, some nucleoside analogs exhibit poor membrane permeability and are poorly soluble in water, thus, limiting their ability to penetrate cells.
2. Attack by Deaminases
A second problem associated with nucleosides analogs having an amine group on the base (i.e., adenine or cytosine) is deamination by host deaminases. For example, deaminases convert araA into arahypoxanthine, which has little antiviral effect.
3. Dependence on Host or Viral Enzymes for Phosphorylation
Nucleoside analogs are dependent on host or viral enzymes for the phosphorylation into the active anti-metabolites to occur. In the absence, or low activity of these enzymes, the nucleoside is poorly converted into the bioactive form.
Administration of the phosphorylated form, i.e., the nucleotide analog, could bypass one or more of the phosphorylation steps performed by the cell, a step in the conversion of the nucleoside into the bioactive form. In addition, nucleotide analogs are structurally and metabolically closer to the therapeutically active phosphorylated form(s). However, the phosphate group is highly charged, which makes the nucleotide analog less membrane-permeable than the nucleoside analog. Secondly, host enzymes, such as phosphatases, hydrolyze the nucleotide analog back to the nucleoside analog, which then must undergo re-phosphorylation. For these reasons, nucleotide analogs are little used in pharmacology.
4. Toxic Effects
When administered to patients, nucleoside analogs have shown toxicity to liver, bone marrow, and the nervous system. In the case of antiviral therapy, nucleoside analogs have been rarely curative, and the side effects that arise during chronic administration of the drug often cause therapy to be discontinued or altered. In the case of cancer therapy, where intent is to kill the cancer cells, the compound and protocol (dose, method of administration, timing of doses) must be carefully designed and monitored to minimize the damage to non-cancerous tissues.
The following various approaches have been attempted to overcome the membrane permeability problem and improve the therapeutic efficacy of the nucleotide analog with limited success.
Low Molecular Weight Prodrugs
Prodrugs of nucleotide analogs are inactive forms of the nucleotide analog which are converted in vivo into the bioactive form. Low molecular weight prodrugs (molecular weights below 5 kilodaltons) consist of various low molecular weight groups reacted with the oxygen atoms of the phosphate group at the 5' OH position. These low molecular weight groups obscure or eliminate the charged oxygen atoms of the phosphate group, thereby allowing cell uptake to occur. (Lefebvre I, et al. J. Med Chem, 1995; 38:3941-3950, incorporated herein by reference). Once inside the cell, these multiple groups must be removed before any antiviral activity is achieved. McGuigan (McGuigan et al, WO 90/05736 Publ. Nov. 23, 1898) synthesized a series of prodrugs of AZT substituted with di and tri ester phosphonate alkyl chains (C1-C18). However, these derivatives were poorly active.
Different Types of Linkages between the Phosphate and the Nucleoside Analog
Phosphonate nucleotide analog prodrugs have a phosphorus atom (--P--) firmly attached to the glycoside portion of the nucleoside analog by a --P--C-- linkage rather than with a --P--O--C-- diester linkage. Though stable to phosphatases, phosphonate nucleotide analogs are highly charged, poorly absorbed after oral administration, and are poorly membrane permeable. Depending on the linking groups used, the cleavage of the --P--C-- linkage can produce toxic intermediates (Krise et al (Krise J P, Stella V. J., Prodrugs of phosphates, phosphonates and phosphinates, Advanced Drug Delivery Reviews, 1996, 19:287-310, incorporated herein by reference).
Receptor Directed Nucleotide Conjugates
Enriquez et al., (U.S. Pat. No. 5,490,991) overcame the membrane permeability problem by coupling nucleotide analogs to molecules recognized by certain receptors. These conjugates were selectively delivered to and internalized by cells bearing the particular receptor. Such conjugates concentrate the drug in receptor positive cells, for example hepatocytes in the liver, and limit its concentration, and toxic effects in non-receptor bearing cells. (Enriquez et al Bioconjugate Chemistry, 1995, 6:195-202.) However, they are limited by the requirement for a receptor on the cell surface. If a pathogen, e.g. virus, is not confined to receptor positive cells, receptor targeting can have limited effectiveness. Furthermore, certain neoplastic cells can lose receptors upon transformation, and cannot internalize the prodrug.
Polymeric Conjugates
High molecular weight polymers have been attached to nucleoside analogs as an attempt to improve the plasma stability and reduce the toxicity of the nucleoside analog. For example, when natural or synthetic polymers are linked to nucleoside analogs via chemically unstable linkages, such as esters, the linkage is subject to hydrolysis in the plasma, resulting in extracellular the release of the nucleoside analog. After release, the nucleoside is subject to enzymatic degradation in the blood as discussed previously. When Usher et al. (PCT WO 95/00177) coupled dextran to AZT, the conjugate was significantly less effective than the unconjugated AZT.
When highly chemically stable linkages between the polymer and the nucleoside analog are used (e.g., ether linkages), the drug may never be released. Instead, the conjugate can be excreted intact or stored for a long period of time, which can lead to toxicity.
Lipid Carrier Conjugates
Yarvin et al (U.S. Pat. No. 5,149,794) attached various antiviral and antineoplastic agents to lipid carrier in an attempt to enhance the rate at which these agents cross the cell membrane. However, these hydrophobic molecules tend to form micelles, which are rapidly cleared by the liver.
The prodrugs of nucleotide analogs that have been prepared to date lack a combination of plasma stability, intracellular lability (releasability) and therapeutic efficacy. Hence, there is a need for prodrugs of nucleotide analogs that are stable in plasma after administration, are capable of traversing cell membranes and releasing a therapeutically available form of the nucleotide analog intracellularly. There is a need furthermore for prodrugs that are capable of treating viral or cancer based diseases with therapeutic efficacy and reduced toxicity.